Extracellular Vesicle Functionalization Using Oligonucleotide Tethers

Abstract

Provided herein are tethered extracellular vesicles and methods of making tethered extracellular vesicles.

1 . A tethered extracellular vesicle comprising:
 an extracellular vesicle, such as a microvesicle or an exosome, obtained from a living cell, tissue, organ, or organism;   a hydrophobically-modified first oligonucleotide anchored to the extracellular vesicle; and   a second oligonucleotide hybridized to the first oligonucleotide linked to a member of a binding pair, a therapeutic agent, a surface, or a polymer.   
     
     
         2 . The tethered extracellular vesicle of  claim 1 , wherein the second oligonucleotide is linked to a polymer, such as a polyacrylate, a polymethacrylate, a polyacrylamide, a polypeptide, a polymethacrylamide, a polypeptide, a polystyrene, a polyethylene oxide, a poly(organo)phosphazene, a poly-L-lysine, a polyethyleneimine, a poly-d,l-lactide-co-glycolide, or a poly(alkylcyanoacrylate). 
     
     
         3 . The tethered extracellular vesicle of  claim 2 , wherein the polymer has a saturated carbon backbone, is prepared from one or more ethylenically unsaturated monomers, and/or has a PDI of less than 2.0. 
     
     
         4 . The tethered extracellular vesicle of  claim 2 , wherein the polymer is an acrylic polymer and wherein the acrylic polymer optionally comprises pendant poly(ethylene oxide) groups comprising the structure —(O—CH 2 —CH 2 —) n , where n is 100 or less, 20 or less or 10 or less, or has an M n  of 200 or less; zwitterionic groups; or methylsulfinyl terminated alkyl groups. 
     
     
         5 . The tethered extracellular vesicle of  claim 1 , wherein the second oligonucleotide is linked to a biologically active agent, such as a therapeutic agent, or a binding reagent, such as an antibody, an antibody fragment, or an aptamer. 
     
     
         6 . The tethered extracellular vesicle of  claim 5 , wherein the second oligonucleotide is linked to a binding reagent complexed with a biologically active agent, such as a therapeutic agent. 
     
     
         7 . The tethered extracellular vesicle of  claim 1 , wherein the extracellular vesicle comprises a therapeutic agent, such as a therapeutic loaded inside the lumen of the extracellular vesicle or on the membrane surface. 
     
     
         8 . A composition comprising the tethered extracellular vesicle of  claim 1 , and a pharmaceutically-acceptable excipient. 
     
     
         9 . A hydrogel comprising two or more of the tethered extracellular vesicles of  claim 2 , wherein the polymer of the two or more tethered extracellular vesicles is cross-linked with a cross-linker, wherein the polymer optionally comprises a saturated carbon backbone and/or is functionalized with poly(ethylene oxide)-containing groups, and the cross-linker comprises poly(ethylene oxide) 
     
     
         10 . The hydrogel of  claim 9 , comprising a biologically active agent, such as a therapeutic agent, such as a therapeutic loaded inside the lumen of the extracellular vesicle or on the membrane surface and/or a biologically active agent, such as a therapeutic agent, tethered to the extracellular vesicle by attachment to, or complexing with the hydrophobically-modified oligonucleotide. 
     
     
         11 . A tethered extracellular vesicle comprising:
 an extracellular vesicle, such as a microvesicle or an exosome, obtained from a living cell, tissue, organ, or organism; and   a hydrophobically-modified oligonucleotide anchored to the extracellular vesicle and linked to a polymer.   
     
     
         12 . The tethered extracellular vesicle of  claim 11 , wherein the polymer is a polyacrylate, a polymethacrylate, a polyacrylamide, a polymethacrylamide, a polypeptide, a polystyrene, a polyethylene oxide, a poly(organo)phosphazene, a poly-L-lysine, a polyethyleneimine, a poly-d,l-lactide-co-glycolide, or a poly(alkylcyanoacrylate). 
     
     
         13 . The tethered extracellular vesicle of  claim 11 , wherein the polymer has a saturated carbon backbone, is prepared from one or more ethylenically unsaturated monomers, and/or PDI of less than 2.0. 
     
     
         14 . The tethered extracellular vesicle of  claim 11 , wherein the polymer is an acrylic polymer and wherein the acrylic polymer optionally comprises pendant poly(ethylene oxide) groups having the structure —(O—CH 2 —CH 2 —) n , where n is 100 or less, 20 or less or 10 or less, or has an M n  of 200 or less; zwitterionic groups; or methylsulfinyl terminated alkyl groups. 
     
     
         15 . A composition comprising the tethered extracellular vesicle of  claim 11 , and a pharmaceutically-acceptable excipient. 
     
     
         16 . A hydrogel comprising two or more of the tethered extracellular vesicles of  claim 11 , wherein the polymer of the two or more tethered extracellular vesicles is cross-linked with a cross-linker, wherein the polymer optionally comprises a saturated carbon backbone and/or is functionalized with poly(ethylene oxide)-containing groups, and the cross-linker comprises poly(ethylene oxide). 
     
     
         17 . The hydrogel of  claim 16 , comprising a biologically active agent, such as a therapeutic agent, such as a therapeutic loaded inside the lumen of the extracellular vesicle or on the membrane surface and/or a biologically active agent, such as a therapeutic agent, tethered to the extracellular vesicle by attachment to, or complexing with the hydrophobically-modified oligonucleotide. 
     
     
         18 . A method of making a tethered extracellular vesicle, comprising:
 anchoring a hydrophobically-modified oligonucleotide to an extracellular vesicle, such as a microvesicle or an exosome, obtained from a living cell, tissue, organ, or organism;   hybridizing to the hydrophobically-modified oligonucleotide a second oligonucleotide complementary to the hydrophobically-modified oligonucleotide and linked to a member of a binding pair, a therapeutic agent, a surface, a polymer initiator group, or a polymer; or   anchoring a hydrophobically-modified oligonucleotide comprising a polymer initiator group to the extracellular vesicle, such as a microvesicle or an exosome, obtained from a living cell, tissue, organ, or organism; and   polymerizing a polymer in a polymerization reaction from the polymer initiator group.   
     
     
         19 . (canceled) 
     
     
         20 . The method of  claim 18 , wherein the polymer is prepared from one or more ethylenically unsaturated monomers and/or has a PDI of less than 2.0. 
     
     
         21 . (canceled) 
     
     
         22 . (canceled) 
     
     
         23 . (canceled) 
     
     
         24 . The method of  claim 18 , wherein the polymerization reaction is conducted using controlled radical polymerization, such as by atom transfer radical polymerization (ATRP), such as Activators ReGenerated by Electron Transfer (ARGET) ATRP, Initiators for Continuous Activator Regeneration (ICAR) ATRP, supplemental activator and reducing agent atom transfer radical polymerization (SARA) ATRP, electrochemically-controlled ATRP (e-ATRP), or photoinduced ATRP. 
     
     
         25 - 35 . (canceled)